GSK-3β expressed in a transgenic mouse

ABSTRACT

A transgenic animal in which GSK-3β protein is over-expressed is useful as a model for neurodegenerative disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/GB01/02218 filed on May 18, 2001, which claims priority from UnitedKingdom Patent Application No. GB0012056.8, filed on May 18, 2000. Bothprior applications are incorporated herein by reference in theirentirety.

The present invention relates to animal models for neurodegenerativedisease, in particular for Alzheimer's disease.

BACKGROUND OF THE INVENTION

Alzheimer's disease, (AD), is the most common neurodegenerative diseasein developed countries, and is characterized by progressive memory lossand impairments in language and behavior that ultimately lead to death(Alzheimer, 1911; Yankner, 1996). The cognitive decline in AD isaccompanied by neuronal atrophy and loss mainly in cortex, hippocampus,and amygdala (Gomez-Isla et al., 1997). In addition to a specificpattern of neuronal cell death, AD is characterized by twoneuropathological hallmarks, senile plaques and neurofibrillary tangles(NFTs).

Senile plaques are extracellular deposits of amyloid fibrils made of the39-43 amino acid β-amyloid peptide (AB) often surrounded by dystrophicneurites (Glenner and Wong, 1984; Masters et al., 1985; Selkoe, 1994).

NFTs are intraneuronally generated aggregates of paired helicalfilaments (PHFs) which are assembled from hyperphosphorylated forms ofthe microtubule-associated protein tau (Greenberg et al., 1992;Grundke-Iqbal et al., 1986; Lee et al., 1991; Morishima-Kawashima etal., 1995). NFTs can be found in all brain regions undergoingdegeneration in AD and their spatio-temporal pattern of appearancecorrelates well with that of cell death and symptomatology (Arriagada etal., 1992; Braak and Braak, 1991; Gomez-Isla et al., 1997).

Molecular insights into AD pathogenesis have arisen from genetic studiesin families affected by inherited forms of AD (FAD). These account foronly a small percentage of AD cases but have allowed the identificationof mutations in three different genes that are responsible fortriggering the disease. These genes are the presenilins-1 and -2 (PS-1and PS-2) and the amyloid precursor protein (APP) (Hardy, 1996).Mutations in APP result in increased production of Aβ (Price andSisodia, 1998) while PS-1 and PS-2 mutations favor processing of APPinto the long and most amyloidogenic form of Aβ (Aβ₄₂) (Citron et al.,1997; Duff et al., 1996; Price and Sisodia, 1998; Scheuner et al.,1996). This genetic evidence together with in vitro and in vivo studiesof Aβ induced neurotoxicity point to Aβ formation and/or aggregation asa key event in triggering AD.

Little is known about downstream intracellular effectors that accountfor neuronal dysfunction, although activation of glycogen synthasekinase-3β (GSK-3β) has been proposed.

GSK-3β is a proline directed serine/threonine kinase that was originallyidentified due to its role in glycogen metabolism regulation and that ismost abundant in the CNS (Woodgett, 1990). Apart from being implicatedin insulin and IGF-1 mediated signal transduction, GSK-3β is alsoinvolved in the wnt/wingless signaling pathway as the key enzymeregulating β-catenin stability and, as a consequence, its translocationto the nucleus and its transcriptional activity (Anderton, 1999; Earthet al., 1997).

GSK-3β is one of the best candidate enzymes for generating thehyperphosphorylated tau that is characteristic of PHFs (Lovestone andReynolds, 1997). GSK-3β can be purified from microtubules (Ishiguro etal., 1988) and has been shown to phosphorylate tau in most siteshyperphosphorylated in PHFs both in transfected cells (Lovestone et al.,1994) and in vivo (Hong et al., 1997; Munoz-Montano et al., 1997).Furthermore, GSK-3β accumulates in the cytoplasm of pretangle neuronsand its distribution in brains staged for AD neurofibrillary changes iscoincident with the sequence of development of these changes (Pei etal., 1999; Shiurba et al., 1996).

Exposure of cortical and hippocampal primary neuronal cultures to Aβ hasbeen shown to induce activation of GSK-3β (Takashima et al., 1996), tauhyperphosphorylation (Busciglio et al., 1995; Ferreira et al., 1997;Takashima et al., 1998), and cell death (Busciglio et al., 1995; Estuset al., 1997; Forloni et al., 1993; Loo et al., 1993; Pike et al., 1991;Takashima et al., 1993). Blockade of GSK-3β expression or activity,either by antisense oligonucleotides or by lithium, prevents Aβ inducedneurodegeneration of cortical and hippocampal primary cultures (Alvarezet al., 1999; Takashima et al., 1993).

PS-1 has been shown to directly bind GSK-3β and tau incoimmunoprecipitation experiments from human brain samples (Takashima etal., 1998). Thus, the ability of PS-1 to bring GSK-3β and tau into closeproximity suggests that PS-1 may regulate phosphorylation of tau byGSK-3β. Mutant forms of PS-1 in transfection experiments result inincreased PS-1/GSK-3β association and increased phosphorylation of tau(Takashima et al., 1998). Furthermore, PS-1 has also been shown to forma complex with the GSK-3β substrate β-catenin in transfected cells(Murayama et al., 1998; Yu et f al., 1998) and in vivo Yu et al., 1998;Zhang et al., 1998) and this interaction increases β-catenin stability(Zhang et al., 1998). Pathogenic PS-1 mutations reduce the ability ofPS-1 to stabilize β-catenin, which in turn results in decreasedβ-catenin levels in AD patients with PS-1 mutations (Zhang et al.,1998).

OBJECT OF THIS INVENTION

There is a need for animal models which closely mimic the pathology ofneurodegenerative diseases such as AD, which are vital for theunderstanding of the disease and testing of new therapies.

The present invention sets out to approach this problem of animalmodels.

SUMMARY OF THE INVENTION

The invention provides a transgenic animal model for Alzheimer'sdisease, wherein GSK-3β protein is over-expressed in the animal.

PREFERRED EMBODIMENTS

In particular, the invention provides a transgenic animal model, whereinexpression of the GSK-3β protein is conditional.

Preferably GSK-3β is the only enzyme which is over-expressed, and indeedit is more preferred that GSK-3β is the only protein which isover-expressed. Overexpression of the GSK-3β protein alone surprisinglyproduces a pathology in the transgenic animal that closely mimics AD.Specifically, GSK-3β overexpression results in decreased levels ofnuclear β-catenin, increased phosphorylation of tau protein, neuronalcell death, reactive astrocytosis and microgliosis. The similarpathology of the transgenic model and the natural disease state makesthe model of the present invention highly valuable for disease analysis.

Preferably the animal used in the transgenic studies is a mammal, suchas a mouse, rat or primate. Other suitable animals for use in transgenicstudies are well known in the art.

The present invention also extends to the methods used for production ofthe transgenic animal.

The animal model of the invention is useful in the testing of new drugsor therapies to treat neurodegenerative diseases such as AD. Thereforethe invention extends to a method of identification of a therapy usefulin the treatment of AD, comprising administering the therapy to thetransgenic animal of the invention and monitoring for an effect onpathology or behaviour.

DETAILED EMBODIMENTS OF THE INVENTION

For preference we employ a tet-regulated system in mice. Thetet-regulated system has been used for conditional gene expression ineukaryotic cell systems and mice (Gingrich and Roder, 1998). By usingthis system to drive transgenic expression of a mutated form ofhuntingtin, some of us have recently generated the first conditionalanimal model of a neurodegenerative disease (Yamamoto et al., 2000). Thetet-regulated system can be particularly useful when mimickingpathological conditions since it may be used to circumvent perinatallethality due to toxicity of the transgene, trigger expression of thetransgene only in adult life, and stop transgene expression oncerelevant phenotypic changes have taken place (Kelz et al., 1999;Yamamoto et al., 2000).

Regulation of the system is achieved through the tetracycline-regulatedtransactivator (tTA), a chimeric protein comprised of the tet-repressorDNA-binding domain and the VP16 trans activation domain (Gossen andBujard, 1992). This protein binds specifically to the tetO operatorsequence and induces transcription from an adjacent CMV minimalpromoter. The combination of both tTA and the tetO elements thus allowsfor continual transactivation of a given transgene. Tetracycline and itsanalogues can bind to tTA. When this happens, tTA is prevented frombinding to tetO, and transcription is inhibited.

In this way, we have produced conditional transgenic mice overexpressingGSK-3β in the brain during adulthood while avoiding perinatal lethalitydue to embryonic transgene expression. These mice show destabilizationof β-catenin and hyperphosphorylation of tau in hippocampal neurons, thelatter resulting in pretangle-like somatodendritic localization of tau.Neurons displaying somatodendritic localization of tau often showabnormal morphologies and detachment from surrounding neuropil. Reactiveastrocytosis and microgliosis were indicative of neuronal stress anddeath. This finding was further confirmed by TUNEL staining of dentategyrus granule cells. Overexpression of GSK-3β in cortex and hippocampusleads to decreased levels of nuclear β-catenin, increasedphosphorylation of tau in AD relevant epitopes, neuronal cell death, andreactive astrocytosis and microgliosis. Our results thereforedemonstrate that in vivo overexpression of GSK-3β results inneurodegeneration and suggest that these mice can be used as an animalmodel to study the relevance of GSK-3β deregulation to the pathogenesisof Alzheimer's disease.

EXAMPLE

The present invention is further illustrated by the following example ofour experimental work.

Generation of Injection Fragment

An 8.0 kb Ase I fragment (BitetO) was used for microinjection. Togenerate BitetO, a 1.5 kb Hind III fragment corresponding to XenopusGSK-3B cDNA with an N-terminal MYC epitope was excised from apcDNA3-GSK3 plasmid (Sanchez et al., 2000). This fragment was subclonedinto the pCRII cloning vector (Invitrogen) digested with Hind III. Thecorrect orientation was tested for by Xho I digestion. A 1.5 kb fragmentwas then excised by Nsi I-Not I digestion and subcloned into the PstI-Not I sites of a plasmid containing a bidirectional tetO sequenceflanked by cytomegalovirus (CMV) minimal promotors with lacZ reportersequences (pBI-3, (Baron et al., 1995)). Lastly, the 8.0 kb Ase I BitetOfragment was microinjected into single-cell CBAxC57BL/6 embryos. Foundermice were identified by PCR and confirmed by Southern analysis. Foundermice were then crossed with wild type CBAxC57BL/6 mice and Southernanalysis was performed on F1 progeny to test for multiple insertionevents of the microinjection freagment. All mice reported here resultedfrom a single integration event (data not shown).

COS Cell Transfections

COS-7 cells were maintained in Dulbecco's modified essential medium(DMEM; Gibco BRL) supplemented with 10% (v/v) fetal bovine serum, 2 mMglutamine, 100 units ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin andincubated in 95% air/5% CO2 in a humidified incubator at 37° C. Cells at50-70% confluency in 35 mm diameters dishes were transiently transfectedwith LipofectAMINE (Gibco BRL)/5 μg of DNA according to themanufacturer's recommendations. Cells were harvested and analysed 48 hfollowing transfection.

Animals

Mice were bred at the Centro de Biologia Molecular “Severo Ochoa” animalfacility. Mice were housed 4 per cage with food and water available adlibitum and maintained in a temperature-controlled environment on a12/12 hour light-dark cycle with light onset at 07:00 hours.

Antibodies

The following anti-tau antibodies were used: 7.51 (Novak et al., 1991)(a kind gift of Dr. C. Wischik, MRC, Cambridge, UK), PHF-1 (Greenberg etal., 1992; Otvos et al., 1994) (a kind gift of Dr. P. Davies, AlbertEinstein Coll., Bronx, N.Y., USA), 12E8 (Seubert et al., 1995) (a kindgift of dr. P. Seubert, Athena, San Francisco, Calif., USA), AD2(Buee-Scherrer et al., 1996) (a kind gift of Dr. C. Mourton-Gilles,Montpellier, France). According to the residue numbering of the longesthuman tau isoform of 441 amino acids (Goedert et al., 1989), antibody12E8 reacts with tau when serine 262 is phosphorylated (Seubert et al.,1995). Antibodies PHF-1 and AD2 recognize tau when serines 396 and 404are phosphorylated (Buee-Scherrer et al., 1996; Otvos et al., 1994).Other monoclonal antibodies were: anti-GSK3-β TransductionLaboratories), anti-β-catenin (Transduction Laboratories),anti-β-tubulin (Sigma), anti-β-galactosidase (Promega), anti-myc(Developmental Studies Hybridoma Bank, Iowa, USA), anti-GFAP(PharMingen, Calif., USA), OX42 (a kind gift of Dra. P. Bovolenta,Instituto Cajal, Spain), EDI (Serotec; UK). Antibody raised against thenuclear protein U″snRNP was kindly donated by Dr. J. Ortin (CNB, Madrid,Spain).

Immunohistochemistry

Mice were deeply anesthetized with pentothal and transcardially perfusedwith 4% paraformaldehyde in 0.1 M phosphate buffer during 10 min. Thebrains were postfixed in 4% paraformaldehyde for two hours at roomtemperature and placed in 30% sucrose in PBS for 48 hours at 4° C.Sagittal sections (30 μm) were cut in a freezing microtome and collectedin PBS. Free floating sections were pretreated with 0.3% H₂O₂ in PBS andincubated overnight at 4° C. with primary antibodies: PHF-1 (1/150),AD-2 (1/2000), anti-myc (1/20), anti-GSK-3β (1/500),anti-β-galactosidase (1/5000), anti-GFAP (1/250), OX42 (1/1000) in PBScontaining 0.2% Triton X-100, 10% normal goat serum (GmCO) and 1% BSA(Boehringer-Mannheim). Following three PBS washes, sections were carriedthrough standard avidin-biotin immunohistochemical protocols using anElite Vectastain kit (Vector Laboratories). Chromogen reaction wasperformed with diaminobenzidine (Sigma) and 0.003% H₂O₂ for ten minutes.The sections were mounted on chromalum-coated slides and coverslippedwith Aqua-PolyMount (Polysciences). Omission of the primary antibodyresulted in absence of labeling.

LacZ Staining

LacZ staining was performed as follows. Fresh frozen sections werepostfixed for 10 minutes in 4% paraformaldehyde in Soren's buffer.Slides were then incubated for 1 hour at 30° C. in lacZ stainingsolution (1 mg/ml X-gal (4-chloro-5-bromo-3-indolyl-β-galactosidase,Boehringer Mannheim), 5 mM potassium ferrocyanide, 5 mM potassiumferricyanide and 2 mM MgCl₂ in PBS). After staining, sections wererinsed and dry-mounted.

Tunel Assay

The DNA fragmentation characteristic of apoptosis was detected by TUNELmethod in paraformaldehyde postfixed brains. The TUNEL staining ofvibratome sections was performed by following manufacturer'sinstructions (In situ Cell Death Detection, POD; Boehringer Mannheim).Treatment with Dnase I was used as a positive control.

Subcellular Fractionation

To prepare membrane and cytosolic extracts, tissues were washed withice-cold phosphate-buffered saline and homogenized in a hypotonic buffer(0.25 M sucrose, 20 mM HEPES pH 7.4, 2 mM EGTA, 1 mM PMSF, 10 μg/mlaprotinin, 10 μg/ml leupeptine and 10 μg/ml pepstatine). The homogenate(Total cellular fraction) was clarified by centrifugation at 850×g for15 min at 4° C.; the resulting supernatant was then centrifuged at100.000×g for 1 h at 4° C. to isolate the membrane fraction as a pelletand the cytoplasmatic fraction as the supernatant.

Brain nuclei were sedimented through a 2 M sucrose cushion. Brain areasfrom three animals were homogenized in 0.32 M sucrose, 10 mM Tris-HCl pH7.4, 3 mM MgCl₂, 1 mM DTT, 0.1% Triton X-100, 10 μg/ml aprotinin, 10g/ml leupeptine and 10 μg/ml pepstatin by using a Potter homogenizerprovided with a loosely fitting Teflon pestle. The homogenate wasfiltered through cheese-cloth and centrifuged for 10 min at 1000×g. Thepellet was resuspended in 3 ml of homogenization medium without Tritonand supplemented with 1.9 M sucrose. This preparation was layered over acushion of 2M sucrose (10 ml) and centrifuged at 12,000×g in a HB4 rotor(Sorvall). The pellet was resuspended in 0.5 ml of 0.32 M sucrose. Thepurity of brain nuclei was assessed by light microscopy after crystalviolet staining. In addittion, U2snRNP, a known nuclear protein, wasused as a nuclear marker in Western blot analysis.

Western Blot Analysis

Brains were quickly dissected on an ice-cold plate. Extracts for Westernblot analysis were prepared by homogenizing the brain areas in ice-coldextraction buffer consisting of 20 mM HEPES, pH 7.4, 100 mM NaCl, 20 mMNaP, 1% Triton X-100, 1 mM sodium orthovanadate, 5 mM EDT A, andprotease inhibitors (2 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptinand 10 μg/ml pepstatin). The samples were homogenized and centrifuged at15,000 g for 20 min at 4° C. The resulting supernatant was collected,and protein content was determined by Bradford. Thirty micrograms oftotal protein was electrophoresed on 10% sodium dodecylsulfate-polyacrylamide gel and transferred to a nitrocellulose membrane(Schleicher and Schuell). The experiments were performed using thefollowed primary monoclonal antibodies: anti-GSK3β (1/2000), PHF-1(1/200), AD2 (1/2000), 12E8 (1/200), 7.51 (1/100), anti-MYC (1/100),anti-β-tubulin (1/5000), anti-β-galactosidase (1/5000). The filters wereincubated with the antibody at 4° C. overnight in 5% nonfat dried milk.A secondary goat anti-mouse antibody (1/5000; Gmco) and ECL detectionreagents (Amersham) were used for immunodetection. Quantitation ofimmunoreactivity was performed by densitometric scanning. Statisticalanalysis was performed using Student's t test.

Tissue Processing for Electron Microscopy

For electron microscopy, vibratome sections were used. Onceimmunostained, the sections were post fixed in 2% OsO₄ for 1 h,dehydrated, embedded in araldite and flat-mounted in Formvar coatedslides, using plastic cover-slips. After polymerization, selected areaswere photographed, trimmed, re-embeded in araldite and re-sectioned at 1μm. These semithin sections were re-photographed and resectioned inultrathin sections. The ultrathin sections were observed in a Jeolelectron microscope, without heavy metal staining to avoid artifactualprecipitates.

FIGURES

FIG. 1. Mouse design. A, schematic representation of the BitetOconstruct. This consists of seven copies of the palindromic tet operatorsequence flanked by two CMV promoter sequences in divergentorientations. This bi-directional promoter is followed by a GSK-3β cDNAsequence (encoding a MYC epitope at its 5′end) in one direction andβ-galactosidase (LacZ) sequence including a nuclear localization signal(NLS) in the other. B, COS cells were co-transfected with the plasmidcontaining the BitetO construct and an expression vector coding forhuman tau (lanes 1 to 6), a third plasmid that allows expression of tTAwas added (lanes 3 to 6) either in the absence (lanes 3 and 4) or in thepresence (lanes 5 and 6) of 1 μg/ml tetracycline in the culture medium.Protein extracts were probed with antibodies against GSK-3β, AD-likephosphorylated tau (PHF-1), total tau (7.51) and β-galactosidase(β-Gal). C, Tet/GSK-3β mice are generated by crossing mice expressingtTA under control of the CarnKlIa promoter (tTA) with mice that haveincorporated the BitetO construct in their genome (TetO). The doubletransgenic progeny (Tet/GSK-3β) are expected to express GSK-3βconstitutively in the brain unless tetracycline or analogs are givenorally thus preventing transactivation by tTA.

FIG. 2. Pattern of transgene expression in Tet/GSK-3β mice. A-B, X-galstaining of brain sagittal sections from PO Tet/GSK-3β mice that wereeither drug-naive (A) or born after giving doxycycline to the mother forfive days immediately prior to birth (B). C-E, β-galactosidaseimmunohistochemistry in sagittal sections of adult (3 months) Tet/GSK-3βmouse brain reveals expression in the different neuronal layers of thecortex (C), the hippocampus (D), and in the striatum (E). H,hippocampus; Cx, cortex; St, striatum; 11-VI, cortical layers; cc,corpus callosum; DG, dentate gyrus, Hil; hillus; GP, globus pallidus.Scale bar in B corresponds to 1 mm in A-B. Scale bar in E corresponds to200 μm in C-E.

FIG. 3. Overexpression of GSK-3B in cortex and hippocampus of Tet/GSK-3Bmice. A, Western blot of protein extracts from cortex (lanes 1 and 2),cerebellum (lanes 3 and 4), and hippocampus (lanes 5 and 6) of wild type(lanes 1, 3, and 5) or Tet/GSK-3β (lanes 2, 4, and 6) mice. B, histogramshowing percent increase of GSK-3β levels in Tet/GSK-3β mice. C-E,immunohistochemistry in cortical sections of wild type (C) or Tet/GSK-3β(D and E) mice; performed with antibodies against GSK-3B (C and D) orMYC (E). F-H, immunohistochemistry in hippocampal sections of wild type(F) or Tet/GSK-3β (G and H) mice; performed with an antibody againstGSK-3β (F and G) or MYC (H). I-K, high power magnification of thedentate gyri shown in F-H. Scale bar corresponds to 100 μm in C-E, 200μm in F-H, 60 μm in I-I, and 40 μm in K.

FIG. 4. Effect of GSK-3β overexpression on β-catenin levels and tauphosphorylation. A, Western blot of total cellular, membrane, cytosolicand nuclear preparations from cortex (Cx) and hippocampus (Hipp) of wildtype (Wt) or T et/GSK-3β (TG) mice probed with anti-β-catenin antibody.B, Western blot of protein extracts from cortex (Cx), cerebellum (Cb),and hippocampus (Hipp) of wild type (Wt) or Tet/GSK-3β (TG) mice probedwith PHF-1 and β-Tubulin antibodies. C, Western blot of hippocampalextracts from wild type (Wt), tTA, TetO, or Tet/GSK-3β mice probed withthe indicated antibodies.

FIG. 5. Somatodendtitic localization of tau in Tet/GSK-3β mice. A-D,PHF-1 immunohistochemistry in the dentate gyrus of wild type (A and B)or Tet/GSK-3β (C and D) mice. E-F, immunohistochemistry performed with7.51 antibody in the dentate gyrus of wild type (E) or Tet/GSK-3β (F)mice. Arrow in B indicates faintly stained mossy fibers. Inset in Cshows higher magnification of a PHF-1 immunostained granule cell. Scalebar corresponds to 200 μm in A-D, and 60 μm in E-F.

FIG. 6. Electron microscopy study of PHF-1 positive neurons. A, Electronmicrograph of two neurons from the dentate gyrus of Tet/GSK-3β mousehippocampus. N1: Nucleus of a PHF-I immunopositive neuron exhibitingmost of its perimeter detached from the surrounding neuropil(arrowheads), in addition to diffi1se cytoplasmic immunostaining. N2:nucleus of a PHF-1 immunonegative neuron. B, Another PHF-1immunopositive neuron from the dentate gyrus of the same mouse of 4Ashowing PHF-1 reaction product in patches and associated with roughendoplasmic reticulum. N Unlabeled nucleus. C, High magnification of theframed portion in FIG. 4B, showing the patches of reaction product(arrows) and the labeling of the rough endoplasmic reticulum. No heavymetal staining was performed. Calibration bar corresponds to 0.5 μm inall panels.

FIG. 7. Neuronal death and reactive gliosis in Tet/GSK-3β mice. A-B,TUNEL staining of the dentate gyrus of wild type (A) or Tet/GSK-3β (B)mice. Arrows indicate TUNEL positive nuclei. C-D, GFAPimmunohistochemistry in the dentate gyrus of wild type (C) or Tet/GSK-3β(D) mice. E, electron micrograph of the dentate gyrus of Tet/GSK-3βmouse hippocampus showing a hypertrophic astrocytic process surroundinga PHF-1 immunostained neuron. N: nucleus. Asterisk: diffuse cytoplasmicimmunostaining. Black star: hypertrophic astrocytic process. Inset: highmagnification of the astrocytic process showing characteristic bundlesof glial intermidiate filaments. F, OX-42 immunostaining of the dentategyrus of a Tet/GSK-3β mouse. Arrows indicate fine immunoreactivemicroglial processes. Arrowheads indicate immunostained reactive cellbodies. SM, stratum moleculare; SG stratum granulare; H, hillus. Scalebars correspond to 50 μm in A-B, 60 μm in C-D, 0.5 μm in E, and 30 μm inF.

MOUSE CONSTRUCT

We generated a plasmid (Biteto) carrying the bi-directional tetresponsive promoter (Baron et al., 1995) followed by both a GSK-3β cDNA(encoding a MYC epitope at its 5′ terminus) in one direction and, in theother direction, a cDNA encoding β-galactosidase (β-gal) fused to anuclear localization signal (FIG. 1A). This plasmid was assayed intransfection experiments performed in COS cells (FIG. 1B). This plasmidby itself or cotransfected with an expression vector coding for tau(FIG. 1B, lanes 1 and 2) had no effect on GSK-3β levels as evidenced byWestern blot with an antibody against GSK-3β. When cotransfected with aplasmid that allows expression of tTA (FIG. 1B, lanes 3 and 4) a markedincrease in GSK-3β levels was evident. This resulted in increasedphosphorylation of tau as evidenced by Western blot with the PHF-1antibody that recognizes a PHF tau phosphorylation epitope. Whentetracycline was present (FIG. 1B, lanes 5 and 6), trans activation ofGSK-3β was abolished. These experiments therefore demonstrateconditional expression of GSK-3β from the BitetO construct (FIG. 1A).

The BitetO construct was then microinjected into oocytes and the fiveresulting transgenic mouse lines were generically designated TetO (FIG.1C). In the tTA mouse lines, the tTA transgene is under the control ofthe Calciumlcalmodulin kinase lIa promoter (CamKlIa-tTA lines E and B)(Mayford et al., 1996). These tTA lines were chosen to allow forrestricted, conditional expression in the CNS, with particularly highexpression in the forebrain (Mayford et al., 1996; Yamamoto et al.,2000). When the TetO mice are crossed with tTA mice, the resultingdouble transgenic progeny (designated Tet/GSK-3β) are expected toconstitutively express both transgenes (FIG. 1C). This expressionhowever can be abolished in the presence of tetracycline or itsanalogues.

Our previous experience in generating conditional transgenic mice withthe tet-regulated system indicates that the genomic site of insertionand/or copy number of the tetO construct influences the final patternand level of transactivation by tTA. The β-Gal reporter sequence in theBitetO construct permits quick analysis of the pattern of transgeneexpression in the double transgenic mice either by X-Gal staining or byimmunohistochemistry against β-Gal, and furthermore, allows for testingthe efficacy of transgene silencing by tetracycline. We took advantageof this to decide which TetO mouse lines were more suitable for ourstudy.

Characterization of Different Tet/GSK-3β Mouse Lines

When the five TetO lines were bred with tTA lines, three of them showedβ-Gal expression only in the striatum (data not shown). The tworemaining TetO lines (lines G6 and G7) were specially suitable for ourstudy since they exhibited high levels of transgene expression in brainregions relevant to AD such as cortex and hippocampus and we havetherefore focused the rest of the study on lines G6 and G7. These twolines transactivate β-Gal in a spatial pattern very similar to that ofendogenous CamKllcα, with expression evident in cortex, hippocampus,striatum and amygdala (FIG. 2). Immunohistochemistry in brain sectionsof adult Tet/GSK-3β mice shows β-Gal expression in the differentneuronal layers of the cortex, the different fields of the hippocampus(including subiculum, CA1, CA2, CA3, and dentate gyrus), and in the 1.1striatum (FIGS. 2C-E). No β-Gal expression was detected in other brainregions such as globus pallidus, thalamus, brainstem and cerebellum(FIGS. 2A, 2E, 3A, and not shown). A similar pattern and level oftransgenic expression was obtained when either CamKIIα-tTA line (E or B)was combined with one TetO line (G6 or G7). In this study we have usedeach combination interchangeably and thus henceforth we will use theterms tTA and TetO for single transgenic mice, and Tet/GSK-3B for thedouble transgenic animals.

Tet/GSK-3B mice were viable and fertile and appeared normal withoutpharmacological intervention to suppress transgene expression. Thisseemed to contradict the previously postulated toxicity of increasedGSK-3β expression in brain (Brownlees et al., 1997). However,heterozygote crosses between tTA and TetO mice did not yield theexpected frequency of 25% for each genotype (wild type, tTA, TetO, andTet/GSK-3β). The Tet/GSK-3β mice were underrepresented (14%, n=401).This might be indicative of lethality due to embryonic overexpression ofGSK-3β in Tet/GSK-3β mice.

We had previously observed perinatal transgene expression and lethalityin our CamKIIα-tTA driven animal model of Huntington's disease (HD94)(Yamamoto et al., 2000). In the case of HD94 mice, if pregnant mice aregiven the tetracycline analog doxycycline (2 mg/ml) in the drinkingwater ad libitum from E15 to birth, only postnatal transgenic expressiontakes place and the frequency of the four expected genotypes is restoredto 25%. We thus decided to apply the same program of perinataldoxycycline treatment to the Tet/GSK-3B mice. We found that at PO, nontreated mice show X-gal staining in the forebrain while staining wasabsent in treated mice (FIGS. 2A and B). This demonstrates thattransgene expression in Tet/GSK-3β mice begins during embryonic life andthat can be inhibited with doxycycline. As expected, prenataldoxycycline treatment normalized to 25% the frequency of Tet/GSK-3β miceand thus, to maximize yield of double transgenic mice in litters, theperinatal doxycycline treatment was routinely employed.

Tet/GSK-3β mice overexpress GSK-3β in cortex and hippocampus. We thenconfirmed by Western blot analysis that the brain regions that showβ-Gal expression also display increased levels of GSK-3β. Probingprotein extracts with an anti-MYC antibody demonstrated that the highestlevel of transgenic GSK-3β expression takes place in the hippocampus,followed by the cortex (FIG. 3A) while little expression could bedetected in the striatum (not shown). Accordingly, probing extracts from3 month old mice with an antibody raised against GSK-3β (FIGS. 3A and B)we observed a significant (p<0.02) 40+/−12.4% increase in GSK-3β levelsin the hippocampus of Tet/GSK-3β mice with respect to wild type mice.Cortical extracts also showed increased (17+/−5%) levels of GSK-3β inTet/GSK-3β mice. No differences in GSK-3β levels were found in thestriatum (not shown) or in non forebrain regions such as cerebellum(FIG. 3B). We then monitored at ages ranging from 1 to 12 months theincrease in GSK-3β that takes place in the hippocampus and cortex ofTet/GSK-3β mice. The level of overexpression was similar at all testedages (not shown) and the rest of expreiments in the present study wereperformed on adult mice at ages between 2.5 and 6 months.

To gain insight into which cell populations are overexpressing GSK-3β,we performed immunohistochemistry with both anti-MYC and anti-GSK-3βantibodies. In the cortex, increased immunoreactivity (IR) for GSK-3βwas found in layer II and III pyramidal cortical neurons (FIGS. 3C-E)and in lamina VI neurons adjacent to the corpus callosum (not shown).

In the hippocampus, overexpression of GSK-3β was evident in all regions(subiculum, CA1, CA2, CA3, and dentate gyrus) with the dentate gyrus andCA2 displaying the most prevalent increase (FIGS. 3F-H). The dentategyrus of wild type mice showed very weak IR for GSK-3β (FIGS. 3F and 3I)while every neuron in the dentate gyrus of Tet/GSK-3β mice overexpressedGSK-3β (FIGS. 3G and 3I). Some of these neurons showed a remarkably highstaining with both anti-GSK-3β and anti-MYC antibodies (FIGS. 3I-K) andoften exhibited abnormal morphologies such as shrunk cell bodies (notshown). In CA2 pyramidal neurons, a prominent staining in both cellbodies and dendrites was seen in Tet/GSK-3β mice (FIGS. 3G-H).

We next analyzed by Western blot, the effect of GSK-3β overexpression onits AD related substrates β-catenin and tau. β-catenin is a component ofcell-cell adherent junctions but also associates with HMG-boxtranscription factors of the Tcf/LEF family and promotes transcriptionof target genes. GSK-3β is the key enzyme regulating β-cateninstabilization and subsequent nuclear translocation (Anderton, 1999;Barth et al., 1997).

We first analyzed the levels of β-catenin in total cortical andhippocampal extracts (FIG. 4A). No differences were found between wildtype and Tet/GSK-3β mice. We then analyzed β-catenin levels in differentcellular compartments. As can be seen in FIG. 4A, no changes wereobserved in β-catenin levels in membrane or cytosolic extracts fromeither cortex or hippocampus. When nuclear extracts were analyzed, nosignificant differences were observed in β-catenin levels in cortex.However, in hippocampus, we observed a significant (p<0.05, n=6) 35+/−8%reduction in nuclear β-catenin levels of Tet/GSK-3˜mice compared to wildtype littermates. This decrease in nuclear β-catenin was also evident byimmuno-electron microscopy in the dentate gyrus of Tet/GSK-3β mice (notshown).

We next performed Western blots with tau antibodies in those brainregions which show MYC expression (cortex, striatum, and hippocampus) aswell as in the cerebellum. Only the hippocampus showed increased levelsof tau phosphorylation as detected by the PHF-1 antibody (FIG. 4B andnot shown). The increase in AD-like phosphorylation of tau detected withthe PHF-1 antibody was reproduced using the AD2 antibody raised againstthe same phosphoepitope of tau (FIG. 4C). The increase in PHF-1 and AD2IR in Tet/GSK-3β mice is not due to altered levels of total tau since noincrease was observed with the phosphorylation independent tau antibody7.51 that recognizes all tau isoforms. Furthermore, phosphorylation atserine 262 which is not adjacent to a proline residue and which has beenshown to be independent of GSK-3β in vivo (Munoz-Montano et al., 1997)is not affected in Tet/GSK-3β mice as detected by the 12E8 antibody(FIG. 4C).

We compared tau phosphorylation and transgenic protein expression in thefour possible genotypes (wild type, tTA, TetO, and Tet/GSK-3β) (FIG.4C). Only Tet/GSK-3β mice showed β-Gal expression and increased levelsof GSK-3β, and PHF-1 and AD2 tau, therefore demonstrating thattransgenic expression and subsequent effects in Tet/GSK-3β mice were dueto transactivation by tTA of the BitetO construct and not due to leakageof the latter in the TetO mice.

Somatodendritic Localization of AD-Like Hyperphosphorylated Tau

We analyzed by immunohistochemistry which hippocampal neuronalpopulations exhibit the increase in PHF-1 IR observed by Western blot.Increased PHF-1, immunostaining was most evident in the dentate gyrus(FIG. 5). In wild type mice, granule cells of the dentate gyrus show nodetectable PHF-1 IR (FIG. 5A), although some staining could be detectedin the mossy fibers projecting to CA3 (FIG. 5B). Tet/GSK-3B mice show amarked increase in the staining of mossy fibers (FIG. 5D) and,interestingly, most granule cells show strong somatodendritic PHF-1immunostaining, thus resembling the pretangle stage of ADneurofibrillary degeneration (FIG. 5C).

Phosphorylation of tau by GSK-3β decreases the affinity of tau formicrotubules in vitro and in transfected cells (Lovestone et al., 1996).This may explain in part the somatodendritic staining found with thePHF-1 antibody. To test this, we performed immunohistochemistry with7.51, an antibody raised against the tubulin binding domain of tau andthat therefore recognizes tau only when it is not bound to microtubules.Interestingly, the 7.51 antibody stained somas of Tet/GSK-3β but notwild type dentate gyrus granule cells (FIG. 5F). The morphology of 7.51stained cells was very similar to that observed with PHF-1 antibody (seeinset in FIG. 5C).

Strong somatodendritic immunostaining of hyperphosphorylated tau mayalso be indicative of aberrant aggregated forms of tau such as PHFs.Thioflavine-S staining in AD brains reveals both neurofibrillary tanglesand amyloid plaques. We therefore performed Thioflavine-S staining inbrain sections of Tet/GSK-3β mice. No Thioflavine-S fluorescence wasdetected, either in the granule cells of the dentate gyrus or in anyother brain region, indicating the absence of PHF bundles and of β-sheetprotein aggregates. The lack of Thioflavine-S fluorescence could stillbe compatible with the existence of few, short PHFs, thus representinginitial steps of neurofibrillary degeneration.

To analyze this possibility, we then studied by electron microscopyTet/GSK-3β granule cells since they show strong somatodendriticimmunolabeling for PHF-1. A diffuse reaction product was present in theperikaryon of these neurons (FIGS. 6A-C), although in some cases we alsoobserved patches of dark reaction product (FIG. 6C). However, PHFs werenot observed either in the dark reaction product patches or in otherportion of the diffuse immunolabeled cytoplasm. Interestingly,immunolabeled material was often seen along the cytoplasmic face of therough endoplasmic reticulum (RER) cisternae, and the above mentioneddark stained patches were, in some occasions, in close proximity ofthese labeled RER cisternae (FIG. 6C). Interestingly, Tet/GSK-3β neuronswith diffuse PHF-1 cytoplasmic labeling very frequently appeareddetached from the surrounding neuropil, showing a widened extracellularspace along most of their periphery (FIG. 6A) whereas unlabeled neuronsdid not show any detachment. We also observed no detachment of granulecell neurons of wild type mice.

Neuronal Cell Death and Reactive Gliosis in the Hippocampus ofTet/GSK-3β Mice

Previous studies have demonstrated that GSK-3B is inhibited by the PI3-kinase/PKB survival pathway which prevents apoptosis (Cross et al.,1995; Cross et al., 1994; Hurel et al., 1996; Saito et al., 1994). This,together with the observation that destabilization of β-catenin bymutations in PS-1 potentiate neuronal apoptosis (Zhang et at., 1998),prompted us to explore whether apoptosis was taking place in Tet/GSK-3βmice as a consequence of the overexpression of GSK-3β.

Different neuronal populations of Tet/GSK-3β mice showed TUNEL labelingthat was absent in wild type mice. This was observed mainly in thegranule cells of the dentate, gyrus (up to 5 labeled granule cells per30 μm section of Tet/GSK-3β dentate gyrus versus no labeling in wildtype dentate gyrus. FIGS. 4A and B). Some TUNEL positive cells were alsoobserved adjacent to the corpus callosum in cortical lamina VI ofTet/GSK-3β mice (not shown).

We next tested whether the neuronal alterations and/or death triggeredby overexpression of GSK-3β in Tet/GSK-3β mice was accompanied by glialalterations such as reactive astrocytosis and microgliosis.Immunohistochemistry performed with an antibody raised against glialfibrillary acidic protein (GF AP) revealed reactive astrocytosis indifferent brain regions. Coincident with TUNEL labeling, GF AP stainingwas most prevalent in the dentate gyrus and in deep cortical layers(FIGS. 7C and D, and not shown). Electron microscopy studies confirmedthe presence of highly activated astrocytic processes, full of glialintermediate filaments, in the dentate gyrus of the Tet/GSK-3β mice.These were often found surrounding PHF-1 IR neurons (FIG. 7E).

To test whether microgliosis was taking place in the hippocampus ofTet/GSK-3β mice we performed immunohistochemistry with OX42, LN-3, andED1 antibodies. Similar results were obtained with all of these threeantibodies (FIG. 7F shows OX-42 immunohistochemistry). When compared towild type hippocampal sections, an increase in fine microglial processeswas found in the stratum granulare of Tet/GSK-3β mice. Furthermoreimmunostained cell bodies corresponding to reactive microglia were foundonly in Tet/GSK-3β mice, mainly in the stratum moleculare of thehippocampus (arrowheads in 7F).

DISCUSSION

By using a conditional transgenic approach here we show that in vivooverexpression of GSK-3β results in neurodegeneration. Conditionaltransgenic mice overexpressing GSK-3β also mimic different biochemicaland cellular aspects of AD such as B-catenin destabilization andpretangle-like somatodendritic localization of hyperphosphorylated tau.Our results therefore support the hypothesis that deregulation of GSK-3βmight be a critical event in the pathogenesis of AD and raise thepossibility that these mice may serve as a useful animal model to studysome aspects of this pathology.

GSK-3β is active during animal development as a component of the Wntsignaling pathway and plays an important role in cell-fate decisions andpattern formation (Bourouis et al., 1990; Ruel et al., 1993; Siegfriedet al., 1992; Siegfried et al., 1994). Accordingly, the ability oflithium to inhibit GSK-3B has been suggested to account for itsteratogenic effects (Klein and Melton, 1996; Stambolic et al., 1996).Apart from their well established roles in early development, Wntsignaling and GSK-3β have been-shown to participate in postnatalcerebellar granule cell synaptogenesis (Hall et al., 2000; Lucas andSalinas, 1997). Toxicity of GSK-3β overexpression during embryonic andpostnatal development of the CNS may explain why Brownlees andcollaborators were unable to generate transgenic mice with detectableoverexpression of GSK-3β even with neuronal specific promoters. Thisprompted these authors to suggest the use of tightly controlledinducible expression systems (Brownlees et al., 1997). In our case, wealso find that a fraction of double transgenic mice die perinatally andthat this can be rescued by silencing transgene expression duringembryonic life. However, some mice can survive without pharmacologicalintervention. There are at least two reasons why this may happen. First,the promoter that we use (CamKlIα) has a more restricted pattern ofexpression than the one employed by Brownilees and collaborators (NF-L).Second, in our binary system the transgene by itself is silent. Thisavoids lethality of founders due to the toxicity of the transgene.Subsequent breeding with tTA mice allows to select those doubletransgenic descendants with a genetic background permissive for theembryonic overexpression of the transgene.

Somatodendritic accumulation of hyperphosphorylated tau is an earlyevent in the evolution of AD neurofibrillary degeneration (Braak et al.,1994). Pretangle-like immunostaining of tau is found in transgenic miceoverexpressing different isoforms of tau (Brion et al., 1999; Gotz etal., 1995; Ishihara et al., 1999; Spittaels et al., 1999).Somatodendritic localization of tau in these mice might be due tosaturation of the tau binding capacity of microtubules. The excess oftau is then susceptible to accumulate in soma and undergo ulteriormodifications such as phosphorylation and conformational changes. InTet/GSK-3β mice, hyperphosphorylation and somatodendritic localizationof tau take place without affecting the total level of tau, therefore,in closer resemblance to the situation found in AD and other taupathies.According to the increase in 7.51 immunostaining found in Tet/GSK-3βmice and to previous in vitro studies (Novak et al., 1991), increasedtau phosphorylation in Tet/GSK-3β mice most likely leads to a decreasedaffinity of tau for microtubules and subsequent accumulation of theprotein in the soma.

We find that somatodendritic tau in Tet/GSK-3β mice is often associatedwith the endoplasmic reticulum. Similar results were found in transgenicmice overexpressing the shortest isoform of tau (Brion et al., 1999) andin aged sheep (Nelson et al., 1993). In all of these cases,immunodetection was performed with antibodies that recognizephosphorylation or conformation epitopes found in PHF-tau.Interestingly, PHFs in AD brains are often found arising from theendoplasmic reticulum and other membrane structures (Gray et al., 1987;Metuzals et al., 1988; Papasozomenos, 1989). It is therefore possiblethat, in animal models, association of tau with the endoplasmicreticulum represents an early stage in the formation of neurofibrillarylesions. An additional and compatible explanation for the association oftau with the endoplasmic reticulum might be its interaction with PS-1.PS-1 has been found to bind both tau and GSK-3β inco-immunoprecipitation experiments performed on human brain extracts(Takashima et al., 1998), and PS-1 is located predominantly in theendoplasmic reticulum and in the Golgi apparatus (Selkoe, 1998).

Several mechanisms may account for the neuronal stress and death(revealed by staining of reactive glia and TUNEL) detected in Tet/GSK-3βmice. In view of the effects of GSK-3β overexpression on tauphosphorylation and compartmentalization, a possible mechanism could bethe disorganization of the microtubule cytoskeleton. In this case, as aconsequence of a diminished stabilization of microtubules by tau, adecrease in microtubule content similar to that found in AD brains(Terry, 1998) would be expected in Tet/GSK-3β mice. Additionally, GSK-3βis negatively regulated by the survival pathway involving PI-3 kinase(Cross et al., 1995; Cross et al., 1994; Hurel et al., 1996; Saito etal., 1994) and challenging cultured cortical neurons with trophic factorwithdrawal or with PI-3 kinase inhibitors leads to stimulation of GSK-3βthat results in apoptosis (Hetman et al., 2000; Pap and Cooper, 1998).Finally, decreased β-catenin mediated transcription has been shown topotentiate neuronal apoptosis in primary neuronal cultures exposed toβ-amyloid (Zhang et al., 1998) or transfected with mutant PS-1 (Weihl etal., 1999). Little is known about the target genes transactivated byβ-catenin which are responsible for the increased susceptibility toapoptosis. Tet/GSK-3β mice can be a useful system to identify such genesby differential display or DNA-micro array approaches.

Substantial progress has been made during the last few years towards thegeneration of transgenic mouse models of AD, particularly in regard tothe β-amyloid toxic cascade and plaque formation (Price and Sisodia,1998). Sequential improvements have been achieved by generating micewith higher expression levels of mutated forms of APP and by breedingthem with mutant PS-1 transgenic mice that favor APP processing intoAβ42 (Guenette and Tanzi, 1999). However, if GSK-3β deregulation is akey event in the pathogenesis of AD, Tet/GSK-3β mice may constitute analternative and/or complementary mouse model of AD.

Most of the effort made to date in transgenic models of AD has focusedon mimicking the neuropathological hallmarks of AD. This may requireexcessively artificial modifications to reproduce within the life-spanof a mouse, something that is formed over many years in a human.Alternatively, it may simply not be possible to mimick all aspects of ADneuropathology in mice because it requires human specific clues (as isthe case for β-amyloid induced toxicity in vivo (Geula et al., 1998).GSK-3β is an enzyme found at the convergence of the pathways involved inAD-like tau hyperphosphorylation, β-amyloid induced toxicity and PS-1mutations. When compared to already existing mouse models of AD,Tet/GSK-3β mice are unique in the sense that they reproduce downstreamintraneuronal dysfunction that may be ultimately responsible for someaspects of AD. A prediction of this hypothesis would be that GSK-3βlevels (or activity) and substrates should be found altered in ADpatients. Evidence in favor of this has already been reported (Pei etal., 1999; Shiurba et al., 1996).

Neurodegeneration in Tet/GSK-3β mice is in good agreement with theneuroprotective effect of lithium, a relatively specific GSK-3βinhibitor. The neuroprotective effects of lithium have been ascribed toits ability to inhibit GSK-3β (Alvarez et al., 1999; Hetman et al.,2000) and to upregulate Bcl-2 (Chen et al., 1999) and downregulate Baxproteins (Chen and Chuang, 1999) in neurons (Revised in Manji et al,1999]. Tet/GSK-3β, mice are thus a good tool to test the neuroprotectiveeffect of forthcoming GSK-3β specific inhibitors. Furthermore, theirefficacy can be compared with the effect of silencing transgeneexpression by administering tetracycline analogs.

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1. A transgenic mouse whose genome comprises a transgene comprising aDNA sequence encoding glycogen synthase kinase-3β (GSK-3β) operablylinked to a tet-regulatable promoter and genome of the mouse furthercomprising a tetracycline-regulated transactivator (tTA) transgene,wherein GSK-3β protein is over-expressed in the mouse and the mousedevelops neurodegeneration.
 2. The transgenic mouse of claim 1, whereinGSK-3β is the only enzyme which is over-expressed.
 3. The transgenicmouse of claim 1, wherein GSK-3β is the only protein which isover-expressed.
 4. A method of identification of a therapy useful in thetreatment of Alzheimer's disease, comprising administering the therapyto the transgenic mouse of claim 1 and monitoring the mouse for aneffect on pathology or behavior.
 5. The transgenic mouse of claim 1,wherein the tTA transgene is under control of a CamKIIα promoter.
 6. Thetransgenic mouse of claim 1, wherein overexpression of GSK-3β is foundin the hippocampus and cortex, but not in the striatum.
 7. A method oftesting a drug or therapy to treat a neurodegenerative disease,comprising administering the drug or therapy to a transgenic mouse ofclaim 1, and monitoring the transgenic mouse for an effect on pathologyor behavior.
 8. A method of making a transgenic mouse over-expressingGSK-3β, comprising introducing a transgene comprising a DNA sequenceencoding glycogen synthase kinase-3β (GSK-3β) operably linked to atet-regulatable promoter into a mouse oocyte, and crossing a resultingmouse whose genome comprises the transgene with a mouse whose genomecomprises a tTA transgene to produce a transgenic mouse of claim 1.